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Flow cytometry detection of inflammatory markers in glial cells and synaptosomes derived from AD cortex
Author(s) -
Melnik Mikhail,
Bilousova Tina,
Huang Calvin,
Cole Emma,
Head Elizabeth,
Gylys Karen H.
Publication year - 2020
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1002/alz.038338
Subject(s) - microglia , trem2 , flow cytometry , pathology , biology , neuroinflammation , immunocytochemistry , lamp1 , microbiology and biotechnology , inflammation , immunology , medicine , lysosome , biochemistry , enzyme
Abstract Background Previous research has indicated the involvement of nearly all cell types of the brain (i.e. neurons, microglia, etc.) in progression of Alzheimer Disease (AD). In the case of microglia, increased production of neuroinflammatory markers accompanies AD and has been implicated in the loss of synapses and neurons. In mouse models of AD, gene expression of many inflammatory markers is increased in microglial cells, including genetic risk factors LPL and TREM2, while homeostatic markers are diminished. Intact cells from post‐mortem AD tissue were isolated and used to better explore the relationship of inflammatory markers and AD‐specific pathological species. Further, microglial elements have been found to be associated with synaptosomes (resealed axon terminals) and were further explored to determine if specific inflammatory signals vary with amyloid and tau pathology. Method Post‐mortem AD tissue (PMI<6 hours) from late AD patients (Braak V‐VI) and age‐matched controls was enzymatically dissociated, processed and labeled using fluorophore‐conjugated antibodies. The labeled cells were subsequently analyzed by an LSRII flow cytometer using FCS Express v5 software. Synaptosomes were prepared from the same AD patients by homogenizing brain tissue in 0.32 M sucrose with a cocktail of enzyme inhibitors. This preparation, known as P2, then underwent probing using western blotting (10‐20% Tris‐Glycine gel with PVDF membrane) and flow cytometry using the same instrument and software. Result Preliminary results show a significant increase in the number of P2ry12+ (homeostatic) microglia in AD patients relative to controls. Further labeling of microglia with CD11b (myeloid marker) and TGFbeta1 (homeostatic marker) showed no significant difference between AD and controls, suggesting a more complex picture of microglial changes compared to mouse models. Preliminary results showed an increase in inflammatory markers TLR4 and IL‐6 in AD P2 samples relative to controls. Further, TLR4 was increased in individuals with at least one APOE4 allele. Conclusion Microglial activation in AD mouse models differs from human AD. Human AD microglia demonstrate an increased expression of one homeostatic marker (P2ry12) but not another (TGFB1) while mice show a more uniform decrease of these markers. Further, microglial signals associated with synaptosomes suggests they contribute to neurotoxicity and synaptic loss, a parameter highly correlated with dementia.